The development of fluorescent probes for detection of reactive oxygen species, such as superoxide and/or hydroxyl radicals, is a central problem in the field of chemical biology. Reactive oxygen species, such as superoxide and the hydroxyl radical, play a significant role in a variety of diseases, such as inflammatory diseases, and probes which can detect reactive oxygen species (ROS) in serum samples, live tissue explants, cell cultures, and/or in vivo have tremendous potential as medical diagnostics and research tools for the diagnoses of diseases characterized by increased ROS production.
Fluorescent sensors for superoxide and the hydroxyl radical have been investigated. For example, dihydroethydium (DHE), the structure of which is shown below, has been used as an ROS probe.

However, DHE has limited applicability due to its spontaneous auto-oxidation, rapid photobleaching, high toxicity, and multiple reaction products with ROS. Further, the lower emission wavelength of DHE makes it difficult to use in vivo. Dihydrorhodamine, another reduced dye that has been investigated for detection of ROS, also suffers from high rates of aerial oxidation, limiting its applications.
Sulfonate ester-based dyes have also been investigated as ROS probes. However, these probes undergo rapid hydrolysis limiting their applications. Moreover, sulfonate ester-based dyes typically involve multistep synthetic procedures which are time consuming and expensive.
There exists a need for ROS probes that do not suffer from the limitations of prior art probes, such as dihydroethydium, dihydrorhodamine, and sulfonate ester-based dyes.
Therefore, it is an object of the invention to provide improved probes for the detection of ROS, and methods of making and using thereof.